Method for detecting erythropoietin (EPO) receptor using anti-human EPO receptor antibodies

ABSTRACT

Herein is reported an antibody that specifically binds to human EPO receptor, wherein the antibody binds to EPO receptor fragment LPGPGGSVDIV (SEQ ID NO: 01) but that does not specifically bind to a protein obtainable from human endothelial cells that has a molecular weight of about 66 kD.

RELATED APPLICATIONS

This application claims the benefit of EP Application No. 11170020.9,filed Jun. 15, 2011. All the teachings of the above-referencedapplication are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 25, 2012, isnamed P4701SeqList.txt and is 5,118 bytes in size.

FIELD OF THE INVENTION

The present invention relates to anti-human EPO receptor antibodies andmethods of using the same.

BACKGROUND

Human erythropoietin (EPO) is a 166-aa glycoprotein which is involved inthe proliferation and differentiation of erythroid progenitor cells.These cellular responses are mediated by the human EPO receptor (EPOreceptor, EPOR), a 508-aa glycoprotein. EPO receptor is a protein of 508amino acid length (Swiss Prot P19235) containing a single transmembranedomain and has been classified as a member of the growth hormonesubfamily of class I cytokine receptors. EPO receptor is described,e.g., in Winkelmann, J. C., et al., Blood 76 (1990) 24-30, and Jones, S.S., et al., Blood 76 (1990) 31-35).

Antibodies against EPO receptor are known from, e.g., D'Andrea, A. D.,Blood 82 (1993) 46-52; Elliott, S., Blood 107 (2006) 1892-1895; Kirkeby,A., J. Neurosci. Methods 164 (2007) 50-58; Miura, O., Arch. Biochem. 306(1993) 200-208; Mayeux, P., et al., J. Biol. Chem. 266 (1991)23380-23385; Westphal. G., et al., Clin. Exp. Med. 2 (2002) 45-52;Elliott, S., et al., J. Immunol. Meth. 352 (2010) 126-139, and EP 1 146056, EP 1 327 681, EP 0 773 962, EP 0 776 370, US 2002/0031806, US2003/0215444, US 2004/0058393, US 2004/0071694, US 2004/0175379, US2005/0227289, US 2005/0244409, US 2006/0018902, U.S. Pat. Nos.6,153,190, 6,998,124, 7,053,184, 7,081,523, WO 1995/005469, WO1996/003438, WO 2000/061637, WO 2004/035603, WO 2005/100403, and WO2010/022924. However, studies investigating the expression andlocalization of EPO receptor in tissue samples produce divergent andoften artifactual results because lack of specificity of knownantibodies against EPO receptor (see Jelkmann, W., et al., Crit. Rev.One. Hematol. 67 (2008) 39-61; Elliott, S., et al., Blood 107 (2006)1892-1895; Jelkmann, W. and Laugsch, M., J. Clin. Oncol. 25 (2007)1627-1628; Kirkeby, A., et al., J. Neurosci. Methods 164 (2007) 50-58;Laugsch, M. et al., Int. J. Cancer 122 (2008) 1005-1011), or it wasreported that studies employed antibodies with questionable specificityand the significance of the observations are controversial (Elliott, S.above).

SUMMARY

It has been found that antibodies as reported herein bind specificallyto human EPO receptor without being cross-reactive to proteins ofsimilar size present on/in endothelial cells allowing for unambiguousdetection results.

One aspect as reported herein is a method for detecting in vitro humanEPO receptor comprising the step of determining in vitro the presence ofhuman EPO receptor in a sample by incubating the sample with an EPOreceptor antibody that specifically binds to human EPO receptor fragmentLPGPGGSVDIV (SEQ ID NO: 01) and thereby detecting in vitro human EPOreceptor, wherein the EPO receptor antibody that specifically binds toEPO receptor fragment LPGPGGSVDIV (SEQ ID NO: 01) does not specificallybind to a protein obtainable from human endothelial cells that has amolecular weight of about 58 kD to about 70 kD.

In one embodiment the method is characterized in that the antibody doesnot specifically bind to a protein obtainable from human endothelialcells that has a molecular weight of about 66 kD.

In one embodiment the method is characterized in that the antibody bindsto the protein obtainable from human endothelial cells with an affinityof 10⁻³M or higher.

In one embodiment the method is characterized in that the antibody is apolyclonal antibody or a monoclonal antibody.

In one embodiment the method is characterized in that the antibody is ahuman, humanized, or chimeric antibody.

In one embodiment the method is characterized in that the antibody is anantibody fragment that binds human EPO receptor.

An aspect as reported herein is an antibody that specifically binds tohuman EPO receptor, characterized in that the antibody binds to humanEPO receptor fragment LPGPGGSVDIV (SEQ ID NO: 01) and does notspecifically bind to a protein obtainable from human endothelial cellsthat has a molecular weight of about 58 kD to about 70 kD.

In one embodiment the antibody is characterized in that it does notspecifically bind to a protein obtainable from human endothelial cellsthat has a molecular weight of about 66 kD.

In one embodiment the antibody is characterized in that it binds to theprotein obtainable from human endothelial cells with an affinity of10⁻³M or higher.

In one embodiment the antibody is characterized in that it is apolyclonal antibody or a monoclonal antibody.

In one embodiment the antibody is characterized in that it is a human,humanized, or chimeric antibody.

In one embodiment the antibody is characterized in that it is anantibody fragment that binds human EPO receptor.

An aspect as reported herein is an antibody that specifically binds tohuman EPO receptor that can be used in a method as reported herein.

An aspect as reported herein is an antibody that specifically binds tohuman EPO receptor for use in a method as reported herein.

In one embodiment the antibody is characterized in that the antibodybinds to EPO receptor fragment LPGPGGSVDIV (SEQ ID NO: 01) and does notspecifically bind to a protein obtainable from human endothelial cellsthat has a molecular weight of about 58 kD to about 70 kD.

In one embodiment the antibody is characterized in that it does notspecifically bind to a protein obtainable from human endothelial cellsthat has a molecular weight of about 66 kD.

In one embodiment the antibody is characterized in that it binds to theprotein obtainable from human endothelial cells with an affinity of10⁻³M or higher.

In one embodiment the antibody is characterized in that it is apolyclonal antibody or a monoclonal antibody.

In one embodiment the antibody is characterized in that it is a human,humanized, or chimeric antibody.

In one embodiment the antibody is characterized in that it is anantibody fragment that binds human EPO receptor.

An aspect as reported herein is the use of an antibody that specificallybinds to human EPO receptor, characterized in that the antibody binds toEPO receptor fragment LPGPGGSVDIV (SEQ ID NO: 01) and does notspecifically bind to a protein obtainable from human endothelial cellsthat has a molecular weight of about 58 kD to about 70 kD for detectinghuman EPO receptor.

In one embodiment the use is characterized in that the antibody does notspecifically bind to a protein obtainable from human endothelial cellsthat has a molecular weight of about 66 kD.

In one embodiment the use is characterized in that the antibody binds tothe protein obtainable from human endothelial cells with an affinity of10⁻³M or higher.

In one embodiment the use is characterized in that the antibody is apolyclonal antibody or a monoclonal antibody.

In one embodiment the use is characterized in that the antibody is ahuman, humanized, or chimeric antibody.

In one embodiment the use is characterized in that the antibody is anantibody fragment that binds human EPO receptor.

An aspect as reported herein is a method for predicting or determiningthe responsiveness of a patient towards a medicament for increasing thenumber of red blood cells comprising

-   -   determining in vitro the presence of human EPO receptor on        cancer cells of the patient by incubating in vitro a sample of        the patient with the EPO receptor antibody as reported herein        and determining in vitro the binding of the antibody to the        sample,        whereby the presence of human EPO receptor on the cancer cells        of the patient is indicative for the responsiveness of the        patient towards a medicament for increasing the number of red        blood cells.

In one embodiment the method is characterized in that the antibody doesnot specifically bind to a protein obtainable from human endothelialcells that has a molecular weight of about 58 kD to about 70 kD, in oneembodiment of about 66 kD.

In one embodiment the method is characterized in that the antibody bindsto the protein obtainable from human endothelial cells with an affinityof 10⁻³M or higher.

In one embodiment the method is characterized in that the antibody is apolyclonal antibody or a monoclonal antibody.

In one embodiment the method is characterized in that the antibody is ahuman, humanized, or chimeric antibody.

In one embodiment the method is characterized in that the antibody is anantibody fragment that binds human EPO receptor.

Herein are reported antibodies that specifically bind to the human EPOreceptor fragment that has the amino acid sequence LPGPGGSVDIV (SEQ IDNO: 01) and that do not bind to human endothelial cells. It has beenfound that such antibodies can be obtained by immunizing an experimentalanimal with the EPO receptor fragment that has the amino acid sequenceLDKWLLPRNPPSEDLPGPGGSVDIV (SEQ ID NO: 02) and thereaftercross-adsorbing, i.e. selecting, the antibodies obtained from theexperimental animal to the EPO receptor fragment with the amino acidsequence LPGPGGSVDIV (SEQ ID NO: 01).

An aspect as reported herein is a method for producing an antibody thatspecifically binds to human EPO receptor comprising the following steps:

-   -   immunizing an animal with a polypeptide comprising the EPO        receptor fragment LDKWLLPRNPPSEDLPGPGGSVDIV (SEQ ID NO: 02), and    -   selecting an antibody that binds to the EPO receptor fragment        LPGPGGSVDIV (SEQ ID NO: 01) and thereby producing an antibody        that specifically binds to human EPO receptor.

In one embodiment the selecting is by cross-adsorbing the antibodiesobtained from the immunized animal to immobilized EPO receptor fragmentof SEQ ID NO: 01.

In one embodiment the method is characterized in that the antibody doesnot specifically bind to a protein obtainable from human endothelialcells that has a molecular weight of about 58 kD to about 70 kD, in oneembodiment of about 66 kD.

In one embodiment the method is characterized in that the antibody bindsto the protein obtainable from human endothelial cells with an affinityof 10⁻³M or higher.

In one embodiment the method is characterized in that the antibody is apolyclonal antibody or a monoclonal antibody.

In one embodiment the method is characterized in that the antibody is ahuman, humanized, or chimeric antibody.

In one embodiment the method is characterized in that the antibody is anantibody fragment that binds human EPO receptor.

An aspect as reported herein is an antibody that specifically binds tohuman EPO receptor, wherein the antibody binds to EPO receptor fragmentLPGPGGSVDIV (SEQ ID NO: 01).

In one embodiment the antibody has an affinity of 10⁻⁷ M or less to thehuman EPO receptor. In one embodiment the antibody has an affinity tothe human EPO receptor of 10⁻⁸ M or less. In one embodiment the antibodyhas an affinity to the human EPO receptor of 5×10⁻⁹ M or less. In oneembodiment the antibody has an affinity to the human EPO receptor of2×10⁻⁹ M or less. In one embodiment the antibody has an affinity to thehuman EPO receptor of about 1.8×10⁻⁹ M. In one embodiment the antibodyhas an affinity to the human EPO receptor of at least 5×10⁻¹⁰ M. In oneembodiment the antibody has an affinity to the human EPO receptor of atleast about 6.3×10⁻¹⁰ M.

In one embodiment of all aspects the antibody is a polyclonal antibodyor a monoclonal antibody.

In one embodiment of all aspects the antibody is a mouse, or rat, orrabbit, or hamster, or sheep, or goat, or chicken, or monkey, or pig, orhuman, or humanized antibody. In one embodiment the antibody is a humanantibody, a humanized antibody, or a chimeric antibody.

In one embodiment the antibody is an antibody fragment that specificallybinds the human EPO receptor.

An aspect as reported herein is an isolated nucleic acid encoding theantibody as reported herein.

An aspect as reported herein is a host cell comprising the nucleic acidencoding the antibody as reported herein.

An aspect as reported herein is a method of producing an antibody asreported herein comprising culturing the host cell as reported herein sothat the antibody is produced.

In one embodiment the method comprises the step of recovering theantibody from the host cell or the cultivation medium.

In one embodiment the host cell is a prokaryotic cell or a eukaryoticcell. In one embodiment the cell is a CHO cell, or a HEK cell, or a BHKcell, or a Sp2/0 cell, or a NS0 cell.

In one embodiment the cell is an E. coli cell or a Bacillus cell.

An aspect as reported herein is a method for producing an antibody thatspecifically binds to the human EPO receptor comprising the followingsteps:

-   -   immunizing an animal with a polypeptide comprising the EPO        receptor fragment LDKWLLPRNPPSEDLPGPGGSVDIV (SEQ ID NO: 02), and    -   selecting an antibody that binds to the EPO receptor fragment        LPGPGGSVDIV (SEQ ID NO: 1), and        thereby producing an antibody that specifically binds to the        human EPO receptor.

In one embodiment the method comprises one or more of the followingadditional steps:

-   -   cultivating a cell comprising a nucleic acid encoding the        antibody that has been selected, and/or    -   recovering the antibody from the cell or the cultivation medium.

An aspect as reported herein is an immunoconjugate comprising theantibody as reported herein and a detectable label or a cytotoxic agent.

An aspect as reported herein is a pharmaceutical formulation comprisingthe antibody as reported herein and optionally a pharmaceuticallyacceptable carrier.

An aspect as reported herein is a diagnostic formulation comprising theantibody as reported herein conjugated to a detectable label.

An aspect as reported herein is the antibody as reported herein for useas a medicament.

An aspect as reported herein is the antibody as reported herein for usein treating anemia.

An aspect as reported herein is the antibody as reported herein for usein changing the number of red blood cells in a patient.

An aspect as reported herein is the use of the antibody as reportedherein in the manufacture of a medicament.

In one embodiment the medicament is for the treatment of anemia.

In one embodiment the medicament is for changing the number of red bloodcells in a patient.

An aspect as reported herein is a method of treating an individualhaving anemia comprising administering to the individual an effectiveamount of the antibody as reported herein for changing/increasing thenumber of red blood cells in the individual.

An aspect as reported herein is a method of changing the number of redblood cells in an individual comprising administering to the individualan effective amount of the antibody as reported herein to change thenumber of red blood cells in the individual.

An aspect as reported herein is a diagnostic kit comprising an antibodyas reported herein.

An aspect as reported herein is a method for the manufacture of adiagnostic kit comprising an antibody as reported herein.

An aspect as reported herein is the use of an antibody as reportedherein for the determination or analysis of the human EPO receptor in ahuman tissue sample.

In one embodiment of all aspects the sample is a lysate of human tissueor human cells.

In one embodiment of all aspects the sample is a section of tissue, or asection of a fresh tissue, or frozen tissue, or a section of frozentissue, or formalin-fixed paraffin embedded tissue, or a section offormalin-fixed paraffin embedded tissue.

In one embodiment of all aspects the analysis is performed byimmunochemistry, immunofluorescence, or immunohistochemistry. In oneembodiment the analysis is performed by Western Blot, or by FACS, or byin-vivo imaging using NIRF or PET.

In one embodiment of all aspects the determination is by incubating thetissue sample with the antibody as reported herein and detecting thebinding of the antibody to the tissue sample.

An aspect as reported herein is a method for predicting or determiningthe responsiveness of a patient towards a medicament for increasing thenumber of red blood cells comprising

-   -   determining in vitro the presence of human EPO receptor on        cancer cells of the patient, and    -   associating the presence of the human EPO receptor on the cancer        cells of the patient with the responsiveness of the patient        towards a medicament for increasing the number of red blood        cells.

An aspect as reported herein is a method for determining the dose of amedicament for increasing the number of red blood cells for treating acancer patient, the method comprising:

-   -   determining in vitro the presence of human EPO receptor on        cancer cells of the patient, and    -   when determining the presence of human EPO receptor deciding to        administer no or a lower dose of a medicament that increases the        number of red blood cells, and    -   when determining the absence of human EPO receptor deciding to        administer a dose of a medicament that increases the number of        red blood cells.

An aspect as reported herein is a method for predicting or determiningthe responsiveness of a patient towards a medicament for increasing thenumber of red blood cells comprising

-   -   determining in vitro the density of human EPO receptor on cancer        cells of the patient,    -   associating the density of human EPO receptors on the cancer        cells of the patient with the responsiveness of the patient        towards a medicament for increasing the number of red blood        cells.

An aspect as reported herein is a method for determining the dose of amedicament for increasing the number of red blood cells for treating acancer patient, the method comprising:

-   -   determining in vitro the presence of human EPO receptor on        cancer cells of the patient, and    -   in the presence of the human EPO receptor deciding to administer        no or a lower dose of a medicament that increases the number of        red blood cells, and    -   in the absence of the human EPO receptor deciding to administer        a normal dose of a medicament that increases the number of red        blood cells.

In one embodiment of all aspects the determining of the presence ofhuman EPO receptor on cancer cells or the determining of the density ofhuman EPO receptor on the cancer cells is by incubating in vitro atissue sample of the patient with an EPO receptor antibody as reportedherein and determining in vitro the binding of the antibody to thesample.

In one embodiment the medicament that increases the number of red bloodcells is erythropoietin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Western Blot analysis of lysates from wt-HELA, HELA-EPOR and UT-7cells.

FIGS. 2A-B Immunocytochemical analysis of wt-HELA and HELA-EPOR cells:A: EPO receptor-GFP fusion protein; B: exemplary antibody as reportedherein.

FIGS. 3A-B Immunohistochemical analysis of wt-HELA and HELA-EPOR cells:A: wild-type HELA cells; B: EPOR-HELA.

FIGS. 4A-B Western Blot analysis of lysates from UT-7, HUVEC (humanumbilical vein endothelial cells) and HMVEC cells (human microvascularendothelial cells).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 01 Fragment of the human EPO receptor that has the amino acidsequence LPGPGGSVDIV.

SEQ ID NO: 02 Fragment of the human EPO receptor that has the amino acidsequence LDKWLLPRNPPSEDLPGPGGSVDIV.

SEQ ID NO: 03 Amino acid sequence of the human EPO receptor precursor.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of non-covalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (likewise by Kd or KD or equilibrium constant). Affinity can bemeasured by common methods known in the art, including those describedherein. When the affinity of polyclonal antibodies is determined,affinity is also denoted as “apparent affinity”. Specific illustrativeand exemplary embodiments for measuring binding affinity are describedin the following.

The terms “anti-human EPO receptor antibody” and “an antibody that bindsto human EPO receptor” refer to an antibody that is capable of bindinghuman EPO receptor of SEQ ID NO: 03 with sufficient affinity such thatthe antibody is useful as a diagnostic and/or therapeutic agent intargeting human EPO receptor. In certain embodiments, an antibody thatbinds to human EPO receptor has a dissociation constant (Kd) of ≦10 nM,≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g. 10⁻⁸ M or less, e.g. from10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certainembodiments, an anti-human EPO receptor antibody binds to an epitope ofhuman EPO receptor that is conserved among human EPO receptor fromdifferent species.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat, E. A. et al., Sequences of Proteins of Immunological Interest,5th ed., Public Health Service, National Institutes of Health, Bethesda,Md. (1991), NIH Publication 91-3242.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody”, “intact antibody”, and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “host cell”, “host cell line”, and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat, E. A. et al., Sequences of Proteins of Immunological Interest,5th ed., Bethesda Md. (1991), NIH Publication 91-3242, Vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR” refers to each of the regionsof an antibody variable domain which are hypervariable in sequenceand/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia, C. and Lesk, A. M., J. Mol. Biol. 196 (1987) 901-917)Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3)occur at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3,31-35B of H1, 50-65 of H2, and 95-102 of H3 (Kabat, E. A. et al.,Sequences of Proteins of Immunological Interest, 5th ed., Public HealthService, National Institutes of Health, Bethesda, Md. (1991), NIHPublication 91-3242). With the exception of CDR1 in VH, CDRs generallycomprise the amino acid residues that form the hypervariable loops. CDRsalso comprise “specificity determining residues,” or “SDRs,” which areresidues that contact antigen. SDRs are contained within regions of theCDRs called abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1,a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at aminoacid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58of H2, and 95-102 of H3. (See Almagro, J. C. and Fransson, J., Front.Biosci. 13 (2008) 1619-1633). Unless otherwise indicated, HVR residuesand other residues in the variable domain (e.g., FR residues) arenumbered herein according to Kabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman, S. et al., J. Chromatogr. B 848 (2007) 79-87.

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-human EPO receptor antibody”refers to one or more nucleic acid molecules encoding antibody heavy andlight chains (or fragments thereof), including such nucleic acidmolecule(s) in a single vector or separate vectors, and such nucleicacid molecule(s) present at one or more locations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 Daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “obtainable from human endothelial cells” denotes in case ofwhole cells the process of paraformaldehyde fixation and in the case oftissue sections the process of deparaffinization followed by epitoperetrieval in citrate buffer at 97° C. for 45 min.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “EPO receptor” refers to any native EPO receptor from anyvertebrate source, including mammals such as primates (e.g. humans) androdents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed human EPO receptor as well as anyform of human EPO receptor that results from processing in the cell. Theterm also encompasses naturally occurring variants of human EPOreceptor, e.g., splice variants or allelic variants. The amino acidsequence of an exemplary human EPO receptor is shown in SEQ ID NO: 03.

The term “treatment” (and grammatical variations thereof such as “treat”or “treating”) refers to clinical intervention in an attempt to alterthe natural course of the individual being treated, and can be performedeither for prophylaxis or during the course of clinical pathology.Desirable effects of treatment include, but are not limited to,preventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, preventing metastasis, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis. In some embodiments, antibodies of theinvention are used to delay development of a disease or to slow theprogression of a disease.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt,T. J. et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y.(2007), page 91) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano, S. et al., J.Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991)624-628).

The term “vector” refers to a nucleic acid molecule capable ofpropagating another nucleic acid to which it is linked. The termincludes the vector as a self-replicating nucleic acid structure as wellas the vector incorporated into the genome of a host cell into which ithas been introduced. Certain vectors are capable of directing theexpression of nucleic acids to which they are operatively linked. Suchvectors are referred to herein as “expression vectors.”

II. Compositions and Methods

In one aspect, the invention is based, in part, on the finding thatantibodies which specifically bind to the human EPO receptor, especiallyto the human EPO receptor fragment of SEQ ID NO: 01, show nocross-reactivity or cross-binding to human epithelial or endothelialcells. In certain embodiments, antibodies that bind to human EPOreceptor are provided. Antibodies of the invention are useful, e.g., forthe diagnosis or treatment of anemia or cancer. Antibodies of theinvention are also useful for the stratification of patients prior tothe administration of a medicament that increases the number of redblood cells, especially prior to the administration of erythropoietin.

A. Exemplary Anti-Human EPO Receptor Antibodies

In one aspect, the invention provides isolated antibodies that bind tohuman EPO receptor. In certain embodiments, an anti-human EPO receptorantibody binds to human EPO receptor fragment LPGPGGSVDIV (EpoR(361-371); SEQ ID NO: 01).

ka kd t/2_(diss) K_(A) K_(D) peptide antibody [1/Ms] [1/sec] [min] [1/M][nM] EpoR GBb 7.9*10⁵    5*10⁻⁴ 23 1.6*10⁹ 0.63 (347-371) EpoR GBb6.4*10⁵ 1.1 *10⁻³ 10 5.7*10⁸ 1.8 (361-371)

In one embodiment the herein provided antibody that binds to human EPOreceptor binds to human EPO receptor fragment 361-371 with a comparableaffinity (comparable Kd-values of the same magnitude) than to EPOreceptor fragment 347-371.

In one embodiment the herein provided antibody that binds to human EPOreceptor has an affinity ratio (Kd-value ratio) for binding to human EPOreceptor fragment 361-371 to binding to human EPO receptor fragment347-371 of less than 10, or less than 5, or about 3.

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g.10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, or e.g. from 10⁻⁹ M to10⁻¹³ M).

In one embodiment, Kd is determined by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of FABs for antigen is measured by equilibrating Fab with aminimal concentration of (¹²⁵I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen, Y. et al., J. Mol.Biol. 293 (1999) 865-881). To establish conditions for the assay,MICROTITER® multi-well plates (Thermo Scientific) are coated overnightwith 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mMsodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovineserum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pMor 26 pM [¹²⁵I]-antigen are mixed with serial dilutions of a Fab ofinterest (e.g., consistent with assessment of the anti-VEGF antibody,Fab-12, in Presta, L. G. et al., Cancer Res. 57 (1997) 4593-4599). TheFab of interest is then incubated overnight; however, the incubation maycontinue for a longer period (e.g., about 65 hours) to ensure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1°A polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150μl/well of scintillant (MICROSCINT-20™; Packard) is added, and theplates are counted on a TOPCOUNT™ gamma counter (Packard) for tenminutes. Concentrations of each Fab that give less than or equal to 20%of maximal binding are chosen for use in competitive binding assays.

According to another embodiment, the Kd value is determined usingsurface plasmon resonance assays using a BIACORE®-2000 or aBIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. withimmobilized antigen CM5 chips at about 10 response units (RU). Briefly,carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flowrate of approximately 25 μl/min. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) is calculated as the ratiok_(off)/k_(on) (see, e.g., Chen, Y. et al., J. Mol. Biol. 293 (1999)865-881). If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmonresonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow equippedspectrophotometer (Aviv Instruments) or a 8000-series SLM-AMINCO™spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson, P. J. etal., Nat. Med. 9 (2003) 129-134. For a review of scFv fragments, see,e.g., Plueckthun, A., In; The Pharmacology of Monoclonal Antibodies,Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York (1994),pp. 269-315; see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 0 404 097; WO1993/01161; Hudson, P. J. et al., Nat. Med. 9 (2003) 129-134; andHolliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448.Triabodies and tetrabodies are also described in Hudson, P. J. et al.,Nat. Med. 9 (2003) 129-134).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison, S. L. et al., Proc. Natl. Acad. Sci. USA 81(1984) 6851-6855). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro, J. C. and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, andare further described, e.g., in Riechmann, I. et al., Nature 332 (1988)323-329; Queen, C. et al., Proc. Natl. Acad. Sci. USA 86 (1989)10029-10033; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and7,087,409; Kashmiri, S. V. et al., Methods 36 (2005) 25-34 (describingSDR (a-CDR) grafting); Padlan, E. A., Mol. Immunol. 28 (1991) 489-498(describing “resurfacing”); Dall'Acqua, W. F. et al., Methods 36 (2005)43-60 (describing “FR shuffling”); and Osbourn, J. et al., Methods 36(2005) 61-68 and Klimka, A. et al., Br. J. Cancer 83 (2000) 252-260(describing the “guided selection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims, M. J. et al., J. Immunol. 151 (1993) 2296-2308;framework regions derived from the consensus sequence of humanantibodies of a particular subgroup of light or heavy chain variableregions (see, e.g., Carter, P. et al., Proc. Natl. Acad. Sci. USA 89(1992) 4285-4289; and Presta, L. G. et al., J. Immunol. 151 (1993)2623-2632); human mature (somatically mutated) framework regions orhuman germline framework regions (see, e.g., Almagro, J. C. andFransson, J., Front. Biosci. 13 (2008) 1619-1633); and framework regionsderived from screening FR libraries (see, e.g., Baca, M. et al., J.Biol. Chem. 272 (1997) 10678-10684 and Rosok, M. J. et al., J. Biol.Chem. 271 (19969 22611-22618).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk, M. A. and vande Winkel, J. G., Curr. Opin. Pharmacol. 5 (2001) 368-374 and Lonberg,N., Curr. Opin. Immunol. 20 (2008) 450-459.

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, N., Nat. Biotech. 23 (2005) 1117-1125.See also, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describingXENOMOUSE™ technology; U.S. Pat. No. 5,770,429 describing HUMAB®technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology,and U.S. Patent Application Publication No. US 2007/0061900, describingVELOCIMOUSE® technology). Human variable regions from intact antibodiesgenerated by such animals may be further modified, e.g., by combiningwith a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described (see, e.g., Kozbor, D.,J. Immunol. 133 (1984) 3001-3005; Brodeur, B. R. et al., MonoclonalAntibody Production Techniques and Applications, Marcel Dekker, Inc.,New York (1987), pp. 51-63; and Boerner, P. et al., J. Immunol. 147(1991) 86-95). Human antibodies generated via human B-cell hybridomatechnology are also described in Li, J. et al., Proc. Natl. Acad. Sci.USA 103 (2006) 3557-3562. Additional methods include those described,for example, in U.S. Pat. No. 7,189,826 (describing production ofmonoclonal human IgM antibodies from hybridoma cell lines) and Ni, J.,Xiandai Mianyixue 26 (2006) 265-268 (describing human-human hybridomas).Human hybridoma technology (Trioma technology) is also described inVollmers, H. P. and Brandlein, S., Histology and Histopathology 20(2005) 927-937 and Vollmers, H. P. and Brandlein, S., Methods andFindings in Experimental and Clinical Pharmacology 27 (2005) 185-191.

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom, H. R. et al., Methods in Molecular Biology 178 (2001) 1-37and further described, e.g., in the McCafferty, J. et al., Nature 348(1990) 552-554; Clackson, T. et al., Nature 352 (1991) 624-628; Marks,J. D. et al., J. Mol. Biol. 222 (1992) 581-597; Marks, J. D. andBradbury, A., Methods in Molecular Biology 248 (2003) 161-175; Sidhu, S.S. et al., J. Mol. Biol. 338 (2004) 299-310; Lee, C. V. et al., J. Mol.Biol. 340 (2004) 1073-1093; Fellouse, F. A., Proc. Natl. Acad. Sci. USA101 (2004) 12467-12472; and Lee, C. V. et al., J. Immunol. Methods 284(2004) 119-132.

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter, G. et al., Ann. Rev.Immunol. 12 (1994) 433-455. Phage typically display antibody fragments,either as single-chain Fv (scFv) fragments or as Fab fragments.Libraries from immunized sources provide high-affinity antibodies to theimmunogen without the requirement of constructing hybridomas.Alternatively, the naive repertoire can be cloned (e.g., from human) toprovide a single source of antibodies to a wide range of non-self andalso self antigens without any immunization as described by Griffiths,A. D. et al., EMBO J. 12 (1993) 725-734. Finally, naive libraries canalso be made synthetically by cloning non-rearranged V-gene segmentsfrom stem cells, and using PCR primers containing random sequence toencode the highly variable CDR3 regions and to accomplish rearrangementin vitro, as described by Hoogenboom, H. R. and Winter, G., J. Mol.Biol. 227 (1992) 381-388. Patent publications describing human antibodyphage libraries include, for example: U.S. Pat. No. 5,750,373, and USPatent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000,2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for human EPO receptor and the other is for any otherantigen. In certain embodiments, bispecific antibodies may bind to twodifferent epitopes of human EPO receptor. Bispecific antibodies may alsobe used to localize cytotoxic agents to cells which express human EPOreceptor. Bispecific antibodies can be prepared as full lengthantibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein, C.and Cuello, A. C., Nature 305 (1983) 537-540, WO 93/08829, andTraunecker, A. et al., EMBO J. 10 (1991) 3655-3659), and “knob-in-hole”engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specificantibodies may also be made by engineering electrostatic steeringeffects for making antibody Fc-heterodimeric molecules (WO 2009/089004);cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat.No. 4,676,980, and Brennan, M. et al., Science 229 (1985) 81-83); usingleucine zippers to produce bi-specific antibodies (see, e.g., Kostelny,S. A. et al., J. Immunol. 148 (1992) 1547-1553; using “diabody”technology for making bispecific antibody fragments (see, e.g.,Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448);and using single-chain Fv (sFv) dimers (see, e.g. Gruber, M et al., J.Immunol. 152 (1994) 5368-5374); and preparing trispecific antibodies asdescribed, e.g., in Tutt, A. et al., J. Immunol. 147 (1991) 60-69).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to human EPOreceptor as well as another, different antigen (see, US 2008/0069820,for example).

The antibody or fragment herein also includes multispecific antibodiesdescribed in WO 2009/080251, WO 2009/080252, WO 2009/080253, WO2009/080254, WO 2010/112193, WO 2010/115589, WO 2010/136172, WO2010/145792, and WO 2010/145793.

7. Antibody Variants

a) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion (see, e.g., Wright, A. and Morrison, S. L., TIBTECH 15 (1997)26-32). The oligosaccharide may include various carbohydrates, e.g.,mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, aswell as a fucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e.g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (EUnumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function(see, e.g., US 2003/0157108; US 2004/0093621). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO 2005/053742; WO 2005/031140; Okazaki, A.et al., J. Mol. Biol. 336 (2004) 1239-1249; Yamane-Ohnuki, N. et al.,Biotech. Bioeng. 87 (2004) 614-622. Examples of cell lines capable ofproducing defucosylated antibodies include Lec13 CHO cells deficient inprotein fucosylation (Ripka, J. et al., Arch. Biochem. Biophys. 249(1986) 533-545; US 2003/0157108; and WO 2004/056312, especially atExample 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.,Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 (2004) 614-622; Kanda, Y.et al., Biotechnol. Bioeng. 94 (2006) 680-688; and WO 2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878; U.S. Pat. No.6,602,684; and US 2005/0123546. Antibody variants with at least onegalactose residue in the oligosaccharide attached to the Fc region arealso provided. Such antibody variants may have improved CDC function.Such antibody variants are described, e.g., in WO 1997/30087; WO1998/58964; and WO 1999/22764.

b) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half-life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch, J. V. and Kinet, J. P., Annu Rev. Immunol. 9(1991) 457-492. Non-limiting examples of in vitro assays to assess ADCCactivity of a molecule of interest is described in U.S. Pat. No.5,500,362 (see, e.g. Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 83(1986) 7059-7063; and Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA82 (1985) 1499-1502); U.S. Pat. No. 5,821,337 (see Bruggemann, M. etal., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactiveassays methods may be employed (see, for example, ACTI™ non-radioactivecytotoxicity assay for flow cytometry (CellTechnology, Inc. MountainView, Calif.; and CytoTox 96® non-radioactive cytotoxicity assay(Promega, Madison, Wis.). Useful effector cells for such assays includeperipheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in an animal model such as thatdisclosed in Clynes, R. et al., Proc. Natl. Acad. Sci. USA 95 (1998)652-656. C1q binding assays may also be carried out to confirm that theantibody is unable to bind C1q and hence lacks CDC activity. See, e.g.,C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. Toassess complement activation, a CDC assay may be performed (see, forexample, Gazzano-Santoro, H. et al., J. Immunol. Methods 202 (1996)163-171; Cragg, M. S. et al., Blood 101 (2003) 1045-1052; and Cragg, M.S, and M. J. Glennie, Blood 103 (2004) 2738-2743). FcRn binding and invivo clearance/half-life determinations can also be performed usingmethods known in the art (see, e.g., Petkova, S. B. et al., Int.Immunol. 18 (2006) 1759-1769).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields, R. L. et al., J. Biol. Chem. 276 (2001) 6591-6604)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie, E. E. et al., J. Immunol. 164(2000) 4178-4184.

Antibodies with increased half-lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer, R. L. et al., J. Immunol. 117 (1976)587-593, and Kim, J. K. et al., J. Immunol. 24 (1994) 2429-2434), aredescribed in US 2005/0014934. Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan, A. R. and Winter, G., Nature 322 (1988) 738-740; U.S.Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

c) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional non-proteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer isattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and non-proteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the non-proteinaceous moiety is a carbonnanotube (Kam, N. W. et al., Proc. Natl. Acad. Sci. USA 102 (2005)11600-11605). The radiation may be of any wavelength, and includes, butis not limited to, wavelengths that do not harm ordinary cells, butwhich heat the non-proteinaceous moiety to a temperature at which cellsproximal to the antibody-non-proteinaceous moiety are killed.

Conjugation methods resulting in linkages which are substantially (ornearly) non-immunogenic are especially suited. Therefore, peptide- (i.e.amide-), sulfide-, (sterically hindered), disulfide-, hydrazone-, orether linkage are especially suited. These linkages are nearlynon-immunogenic and show reasonable stability within serum (see e.g.Senter, P. D., Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO2009/059278; WO 95/17886).

Depending on the biochemical nature of the moiety and the antibodydifferent conjugation strategies are at hand. In case the moiety isnaturally occurring or recombinant of between 50 to 500 amino acids,there are standard procedures in text books describing the chemistry forsynthesis of protein conjugates, which can be easily followed by theskilled artisan (see e.g. Hackenberger, C. P. R., and Schwarzer, D.,Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In one embodimentthe reaction of a maleinimido moiety with a cysteine residue within theantibody or the moiety is used. This is an especially suited couplingchemistry in case e.g. a Fab or Fab′-fragment of an antibody is used.Alternatively in one embodiment coupling to the C-terminal end of theantibody or moiety is performed. C-terminal modification of a protein,e.g. of a Fab-fragment can e.g. be performed as described (Sunbul, M.and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371).

In general site specific reaction and covalent coupling is based ontransforming a natural amino acid into an amino acid with a reactivitywhich is orthogonal to the reactivity of the other functional groupspresent. For example, a specific cysteine within a rare sequence contextcan be enzymatically converted in an aldehyde (see Frese, M. A., andDierks, T., ChemBioChem. 10 (2009) 425-427). It is also possible toobtain a desired amino acid modification by utilizing the specificenzymatic reactivity of certain enzymes with a natural amino acid in agiven sequence context (see, e.g., Taki, M. et al., Prot. Eng. Des. Sel.17 (2004) 119-126; Gautier, A. et al. Chem. Biol. 15 (2008) 128-136; andProtease-catalyzed formation of C—N bonds is used by Bordusa, F.,Highlights in Bioorganic Chemistry (2004) 389-403).

Site specific reaction and covalent coupling can also be achieved by theselective reaction of terminal amino acids with appropriate modifyingreagents.

The reactivity of an N-terminal cysteine with benzonitrils (see Ren, H.et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662) can be used toachieve a site-specific covalent coupling.

Native chemical ligation can also rely on C-terminal cysteine residues(Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology(2009), 22 (Protein Engineering), 65-96).

EP 1 074 563 describes a conjugation method which is based on the fasterreaction of a cysteine within a stretch of negatively charged aminoacids with a cysteine located in a stretch of positively charged aminoacids.

The moiety may also be a synthetic peptide or peptide mimic. In case apolypeptide is chemically synthesized, amino acids with orthogonalchemical reactivity can be incorporated during such synthesis (see e.g.de Graaf, A. J. et al., Bioconjug. Chem. 20 (2009) 1281-1295). Since agreat variety of orthogonal functional groups is at stake and can beintroduced into a synthetic peptide, conjugation of such peptide to alinker is standard chemistry.

In order to obtain a mono-labeled polypeptide the conjugate with 1:1stoichiometry may be separated by chromatography from other conjugationside-products. This procedure can be facilitated by using a dye labeledbinding pair member and a charged linker. By using this kind of labeledand highly negatively charged binding pair member, mono conjugatedpolypeptides are easily separated from non labeled polypeptides andpolypeptides which carry more than one linker, since the difference incharge and molecular weight can be used for separation. The fluorescentdye can be useful for purifying the complex from un-bound components,like a labeled monovalent binder.

In one embodiment the effector moiety is selected from the groupconsisting of a binding moiety, a labeling moiety, and a biologicallyactive moiety.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-human EPO receptor antibodydescribed herein is provided. Such nucleic acid may encode an amino acidsequence comprising the VL and/or an amino acid sequence comprising theVH of the antibody (e.g., the light and/or heavy chains of theantibody). In a further embodiment, one or more vectors (e.g.,expression vectors) comprising such nucleic acid are provided. In afurther embodiment, a host cell comprising such nucleic acid isprovided. In one such embodiment, a host cell comprises (e.g., has beentransformed with): (1) a vector comprising a nucleic acid that encodesan amino acid sequence comprising the VL of the antibody and an aminoacid sequence comprising the VH of the antibody, or (2) a first vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and a second vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VH of the antibody.In one embodiment, the host cell is eukaryotic, e.g. a Chinese HamsterOvary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp2/0 cell). In oneembodiment, a method of making an anti-human EPO receptor antibody isprovided, wherein the method comprises culturing a host cell comprisinga nucleic acid encoding the antibody, as provided above, underconditions suitable for expression of the antibody, and optionallyrecovering the antibody from the host cell (or host cell culturemedium).

For recombinant production of an anti-human EPO receptor antibody,nucleic acid encoding an antibody, e.g., as described above, is isolatedand inserted into one or more vectors for further cloning and/orexpression in a host cell. Such nucleic acid may be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, K. A., In:Methods in Molecular Biology, Vol. 248, Lo, B. K. C. (ed.), HumanaPress, Totowa, N.J. (2003), pp. 245-254, describing expression ofantibody fragments in E. coli). After expression, the antibody may beisolated from the bacterial cell paste in a soluble fraction and can befurther purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern (see Gerngross, T. U., Nat. Biotech. 22 (2004) 1409-1414; andLi, H. et al., Nat. Biotech. 24 (2006) 210-215).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham, F. L. et al., J. Gen Virol. 36(1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4cells as described, e.g., in Mather, J. P., Biol. Reprod. 23 (1980)243-252); monkey kidney cells (CV1); African green monkey kidney cells(VERO-76); human cervical carcinoma cells (HELA); canine kidney cells(MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); humanliver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, asdescribed, e.g., in Mather, J. P. et al., Annals N.Y. Acad. Sci. 383(1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian hostcell lines include Chinese hamster ovary (CHO) cells, including DHFR⁻CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980)4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For areview of certain mammalian host cell lines suitable for antibodyproduction, see, e.g., Yazaki, P. and Wu, A. M., Methods in MolecularBiology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, N.J.(2004), pp. 255-268.

C. Assays

Anti-human EPO receptor antibodies provided herein may be identified,screened for, or characterized for their physical/chemical propertiesand/or biological activities by various assays known in the art.

1. Immunohistochemical Staining Assays

In one aspect, tissue sections are deparaffinized, i.e. the paraffin isremoved, followed by epitope retrieval using e.g. a citrate buffer (asavailable from Vector Laboratories) for treatment at elevatedtemperatures, such as for a treatment for 45 min at 97° C. Afterblocking (e.g. with Protein Block Serum-Free (cat no. X0909, availablefrom DAKO Deutschland GmbH)), tissue sections are incubated with theprimary antibody, e.g. in case of the antibody as reported herein at aconcentration of 127.5 ng/ml for 60 min. Afterwards the bound antibodyis determined, e.g. with the Envision polyclonal rabbit detection kit(DAKO Deutschland GmbH). Finally, specimens are counterstained,dehydrated and mounted.

2. Stratification of Patients by Determining the EPO Receptor Status ofCancer Cells

Sample material from patients is collected either from tissue materialfrom a tumor resection, e.g. performed to treat the cancer disease, orby tumor biopsy. Collected tumor tissue is fixed with formalin andembedded in paraffin according to histological standard procedures.Immunohistochemistry using the anti-EPO receptor antibody as reportedherein is performed on sections of this tumor material (see above andexamples). Histopathological assessment of stained tissue sectionsallows for the determination whether the tumor tissue is EPO receptorpositive or negative. This assessment can be based on a scoring systemwhich takes into account the intensity of the staining of tumor cellsand the number of stained tumor cell in a defined section area.

Assessment of EPO receptor status of the tumor tissue of a patient canbe the basis for determining an optimized treatment of the cancerpatient. This includes prognosis of tumor progression, intensity ofanti-tumor therapies like radiation therapy, chemotherapy, therapy byspecific anti-tumor agents, treatment of chemotherapy or tumordisease-associated anemia, change in dose or administration schedule ofan erythropoiesis stimulating agent (ESA) used to correct the anemiccondition of the patient, discontinuation of the anti-anemic treatmentwith an ESA, change from one particular type of ESA to another type ofESA, switch from anti-anemia treatment with an ESA to transfusions ofblood or separated and concentrated erythrocytes (packed red bloodcells), or a decision to postpone or to refrain from future treatmentswith an ESA.

ESAs are agents which stimulate erythropoiesis e.g. by stimulation ofthe erythropoietin receptor like recombinant human erythropoietins, orepoetins, modified erythropoietins, continuous erythropoietin receptorstimulators, small molecule or peptidic erythropoietin receptoragonists, stabilizers of hypoxia inducible factors etc.

D. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-human EPO receptor antibodiesprovided herein is useful for detecting the presence of human EPOreceptor in a biological sample. The term “detecting” as used hereinencompasses quantitative or qualitative detection. In certainembodiments, a biological sample comprises a cell or tissue, such asPBMCs (peripheral blood monocytic cells), tissue sections or samplesfrom normal or diseased tissues, fresh tissues, frozen tissues,formalin-fixed, paraffin-embedded (FFPE) tissues.

In one embodiment, an anti-human EPO receptor antibody for use in amethod of diagnosis or detection is provided. In a further aspect, amethod of detecting the presence of human EPO receptor in a biologicalsample is provided. In certain embodiments, the method comprisescontacting the biological sample with an anti-human EPO receptorantibody as described herein under conditions permissive for binding ofthe anti-human EPO receptor antibody to human EPO receptor, anddetecting whether a complex is formed between the anti-human EPOreceptor antibody and human EPO receptor. Such method may be an in vitroor in vivo method. In one embodiment, an anti-human EPO receptorantibody is used to select subjects eligible for therapy with arecombinant human EPO (epoetin), hyperglycosylated humanerythropoietins, erythropoietin receptor agonists, erythropoietinmimetic peptides, chemical erythropoietin receptor activating compoundsor other erythropoiesis stimulating agents (ESAs) e.g. where human EPOreceptor is a biomarker for selection and/or stratification of patients,or adjusting the dose used in such therapies.

Exemplary disorders that may be diagnosed using an antibody of theinvention include e.g. cancer or also EPOR status of stem cells—fortissue regeneration.

In certain embodiments, labeled anti-human EPO receptor antibodies areprovided. Labels include, but are not limited to, labels or moietiesthat are detected directly (such as fluorescent, chromophoric,electron-dense, chemiluminescent, and radioactive labels), as well asmoieties, such as enzymes or ligands, that are detected indirectly,e.g., through an enzymatic reaction or molecular interaction. Exemplarylabels include, but are not limited to, the radioisotopes ³²P, ¹⁴C,¹²⁵I, ³H, and ¹³¹I, fluorophores such as rare earth chelates orfluorescein and its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, luceriferases, e.g., firefly luciferase and bacterialluciferase (U.S. Pat. No. 4,737,456), luciferin,2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkalinephosphatase, β-galactosidase, glucoamylase, lysozyme, saccharideoxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like.

E. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-human EPO receptor antibody asdescribed herein are prepared by mixing such antibody having the desireddegree of purity with one or more optional pharmaceutically acceptablecarriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A.(ed.) (1980)), in the form of lyophilized formulations or aqueoussolutions. Pharmaceutically acceptable carriers are generally nontoxicto recipients at the dosages and concentrations employed, and include,but are not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyl dimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinyl pyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude interstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rhuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rhuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO 2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such active ingredients are suitably present in combination inamounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methyl methacrylate) microcapsules, respectively, in colloidaldrug delivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Osol, A. (ed.) (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

F. Therapeutic Methods and Compositions

Any of the anti-human EPO receptor antibodies provided herein may beused in therapeutic methods.

In a further aspect, the invention provides for the use of an anti-humanEPO receptor antibody in the manufacture or preparation of a medicament.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the anti-human EPO receptor antibodies providedherein. In one embodiment, a pharmaceutical formulation comprises any ofthe anti-human EPO receptor antibodies provided herein and apharmaceutically acceptable carrier. In another embodiment, apharmaceutical formulation comprises any of the anti-human EPO receptorantibodies provided herein and at least one additional therapeuticagent, e.g., as described below.

Antibodies of the invention can be used either alone or in combinationwith other agents in a therapy. For instance, an antibody of theinvention may be co-administered with at least one additionaltherapeutic agent.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant. Antibodies of the invention can alsobe used in combination with radiation therapy.

An antibody of the invention (and any additional therapeutic agent) canbe administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.every week or every three weeks (e.g. such that the patient receivesfrom about two to about twenty, or e.g. about six doses of theantibody). An initial higher loading dose, followed by one or more lowerdoses may be administered. However, other dosage regimens may be useful.The progress of this therapy is easily monitored by conventionaltechniques and assays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to an anti-human EPO receptor antibody.

III. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of or in additionto an anti-human EPO receptor antibody.

IV. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1 Generation of Antibodies Directed Against the IntracellularDomain of the Human EPOR

A 25 amino-acid synthetic peptide corresponding to residues 347-371 ofthe mature human erythropoietin receptor (LDKWLLPRNPPSEDLPGPGGSVDIV, SEQID NO: 02) was used as immunogen (corresponds to amino acid residues371-395 of the EPO receptor precursor, SEQ ID NO: 03).

For immunization, peptides were coupled to KLH (keyhole limpethaemocyanine) via a C-terminal cysteine residue. Rabbits were immunizedwith the protein every 4 weeks for 3-5 times. First level screening forspecific antibodies was done by testing on ELISA microtiter platescoated either with protein or biotinylated peptides according toestablished procedures.

Polyclonal sera were precipitated by ammonium sulphate. IgGs wereseparated by DEAE chromatography and further purified by immunoaffinityadsorption on a peptide presenting column containing amino acid residues361-371 of the mature hEPO receptor (SEQ ID NO: 01). IgGs were recoveredfrom the column using propionic acid, pH 2.6. The obtained solution wasadjusted to pH 7.3 using TRIS buffer (2.5 M, pH 8.5). Next, purifiedIgGs were dialyzed against 50 mM potassium phosphate/150 mM KCl bufferfollowed by gel filtration on a Superdex 200 column (GE Healthcare)using the same buffer and finally sterile filtered using a 0.2 μmmembrane filter.

Example 2 Generation of EPO Receptor Expressing HELA Cells

For generating stably transfected HELA cells expressing recombinanthuman EPO receptor (EPOR), cells were transduced with the supernatantfrom HEK 293 cells transiently transfected with a retroviral expressionvector encoding EPO receptor or EPO receptor-eGFP (as fusion protein tothe intracellular C-terminus, Invitrogen) and pVSV-G (an expressionvector encoding the G glycoprotein of the rhabdovirus vesicularstomatitis virus). Two days after transduction the medium was replacedwith fresh supplemented RPMI containing 0.2 mg/ml zeocin.

For transient transfection experiments 8×10⁴ HELA cells were plated oncover slips in a 12-well plate in 1 ml medium using FuGENE Transfectionreagent (Roche Molecular Biochemicals Cat. No. 1815075). In detail, 3 μlof FuGENE 6 were added to 97 μl RPMI 1640 without FCS, incubated for 5min at RT. Thereafter, 1 μg DNA mix was added and incubated for 15 minat RT. Finally, 50 μl of the DNA/FuGENE 6 solution was added to 1 mlcell culture medium containing the cells on cover slips.

Example 3 Generation of EPO Receptor Expressing UT7 Cells

UT-7 cell line is a human factor-dependent erythroleukemic cell line(Human bone marrow acute myeloid leukemia cell line DSMZ: ACC 137),requiring EPO for long-term growth. UT7 cells were maintained in RPMImedium supplemented with L-glutamine (2 mM), non-essential amino acids(1×), sodium pyruvate (1 mM), 10% fetal calf serum and 10 U/ml GM-CSF.Transduced cells (UT7/EPOR) were maintained in the same medium asnon-transduced cells with 25 U/ml GM-CSF instead of 10 U/ml with theaddition of 0.4 mg/ml zeocin. Before each stimulation the cells werestarved by incubation overnight in RPMI media supplemented withL-glutamine (2 mM), non-essential amino acids (1×), sodium pyruvate (1mM) and 0.1% fetal calf serum.

UT-7 cells were transduced with the supernatant from HEK 293 cellstransiently transfected with a retroviral expression vector encoding EPOreceptor and pVSV-G. Two days after transduction the medium was replacedwith fresh supplemented RPMI containing 0.4 mg/ml zeocin and 25 U/mlGM-CSF. After selection a cell line of UT-7 cells stable expressing EPOreceptor on their surface was obtained.

Example 4 SDS-PAGE and Western Blotting

The SDS-PAGE and western blotting were performed according to standardprocedures and the NuPAGE gel system of Invitrogen. The extractscorresponding to different number of cells were loaded in each line of aNuPAGE Novex 4-12% Bis-Tris gel. After gel electrophoresis the proteinswere transferred onto PVDF membranes and incubated with an anti-EPOreceptor antibody obtained in example 1 overnight at 4° C. Afterwashing, the membranes were incubated with a conjugate anti-mouse oranti-rabbit IgG-HRP and developed using ECL reagents (LUMI-Light® PLUSwestern blotting substrate, Roche Diagnostics GmbH, Mannheim, Germany).Results are shown in FIG. 1.

Example 5 BIACORE Analysis

ka kd t/2diss KA KD Chi² peptide antibody [1/Ms] [1/sec] [min] [1/M][nM] RU² EPOR GBb 7.9E+05 5.0E−04 23 1.6E+09 0.63 0.3 (347-371) EpoR GBb6.4E+05 1.1E−03 10 5.7E+08 1.8 0.1 (361-371)

Kinetics of anti-EPO receptor antibody GBb binding to the EPO receptorfragments was determined at 25° C.

Antibody GBb displays nanomolar avidity to the EPO receptor fragment361-371 and nanomolar avidity to EPO receptor fragment 347-371. Theantibody shows a long dissociation constant (t/2 diss).

The antibody GBb was captured to the flow cell of the sensor chip usinggoat anti-rabbit Fcγ antibody followed by perfusion with EPO receptorfragment 347-371 or 361-371, respectively.

Measurements were made on a BIACORE® 3000 instrument at 25° C. inHBS-EP-Buffer, pH 7.4 (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.005%polysorbate 20 (w/v)). 1.0 mg/ml CMD (carboxymethyldextrane) was addedto reduce unspecific binding. The GBb antibody displays nanomolarbinding avidity to the corresponding EPO receptor fragment of SEQ ID NO:01.

Example 6 Immunocytochemistry I

Immunocytochemistry analysis of affinity purified polyclonal antibodydirected against EPO receptor on transiently transfected HELA EPOR cellswere performed as follows: HELA cells cultured on glass coverslips weretransfected to transiently express EPO receptor-GFP fusion protein, PFA(paraformaldehyde) fixed, and stained with 1.0-10 μg/ml purified IgG ofa polyclonal antibody binding to EPO receptor. Bound antibodies weredetected by CY3 goat anti-human IgG secondary antibodies. Specimens wereimaged on a LEICA confocal laser scanning microscope SP2 using 488 nmand 543 nm excitation for Alexa Fluor 488 and CY3 respectively.

Anti-EPO receptor antibody immunoreactivity was found to be closelyco-localized with the green fluorescence of the EPO receptor-GFP fusionprotein. The antibody also recognizes newly synthesized EPO receptorthat is confined to the ER/Golgi region. The lack of any detectablelabeling in non-transfected cells also confirms the high specificity ofthe anti-EPO receptor polyclonal antibodies as reported herein.

Results are shown in FIG. 2.

Example 7 Immunohistochemistry II

Tissue sections of xenograft tissue from either HELA EPOR or wt-HELAcells (neg. for EPOR expression) were deparaffinized followed by epitoperetrieval in citrate buffer (Vector Laboratories) at 97° C. for 45 min.Automated staining was performed on a Labvision instrument. The anti-EPOreceptor antibody was used at 127.5 ng/ml final concentration. Theprimary antibody was detected using the Envision Polyclonal Rabbitdetection kit (DAKO Deutschland GmbH). Images were taken on a ZeissAxiovision Rel 4.8, Microscope (see FIG. 3).

Example 8 Western Blot Analysis on UT-7 Cells, Human Umbilical Vein(HUVEC) and Human Microvascular Endothelial (HMVEC) Cells

Methods as described in Example 4 were used.

Panel A in FIG. 4 shows the immunoreactivity of an antibody binding tothe EPO receptor fragment LDKWLLPRNPPSEDLPGPGGSVDIV (SEQ ID NO: 02).

Panel B in FIG. 4 shows the immunoreactivity of an antibodycross-adsorbed to the EPO receptor fragment LPGPGGSVDIV (SEQ ID NO: 01).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

The invention claimed is:
 1. A method for detecting human erythropoietin(EPO) receptor in vitro comprising determining in vitro the presence ofhuman EPO receptor in a sample by incubating the sample with an EPOreceptor antibody that specifically binds to human EPO receptor fragmentof SEQ ID NO: 01 thereby detecting in vitro human EPO receptor, whereinthe EPO receptor antibody that specifically binds to EPO receptorfragment of SEQ ID NO: 01 does not specifically bind to a proteinobtainable from human endothelial cells that has a molecular weight ofabout 66 kD.
 2. The method according to claim 1, wherein the antibodydoes not specifically bind to a protein obtainable from humanendothelial cells that has a molecular weight of about 58 kD to about 70kD.
 3. The method according to claim 1, wherein the antibody binds to aprotein obtainable from human endothelial cells with an affinity of10⁻³M or higher.
 4. The method according to claim 1, wherein theantibody is a polyclonal antibody or a monoclonal antibody.
 5. Themethod according to claim 1, wherein the antibody is a human, humanized,or chimeric antibody.
 6. The method according to claim 1, wherein theantibody is an antibody fragment that binds human EPO receptor.
 7. Themethod of claim 1, wherein the sample is a human tissue or cell sample.8. The method according to claim 7, wherein the sample is a lysate ofhuman tissue or cells, or a section of human tissue, or a section of afresh human tissue, or frozen human tissue, or a section of frozen humantissue, or formalin-fixed paraffin embedded human tissue, or a sectionof formalin-fixed paraffin embedded human tissue.
 9. The methodaccording to claim 7, wherein analysis is performed by immunochemistry,immunofluorescence or immunohistochemistry.
 10. The method according toclaim 7, wherein analysis is performed by Western Blot.